α-Amylases (α-1,4 glucan-4-glucanohydrolase, EC 3.2.1.1) constitute a group of enzymes which is capable of hydrolyzing starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. Almost all α-amylases studied have a few conserved regions with approximately the same length and spacing. One of these regions resembles the Ca2+ binding site of calmodulin and the others are thought to be necessary for the active centre and/or binding of the substrate.
While the amino acid sequence and thus primary structure of a large number of α-amylases are known, it has proved very difficult to determine the three-dimensional structure of all α-amylases. The three-dimensional structure can be determined by X-ray crystallographic analysis of α-amylase crystals, but it has proven difficult to obtain α-amylase crystals suitable for actually solving the structure.
Until now the three-dimensional structure of only a few α-amylases have been determined at high resolution. These include the structure of the Aspergillus oryzae TAKA α-amylase (Swift et al., 1991), the Aspergillus niger acid amylase (Brady et al, 1991), the structure of pig pancreatic α-amylase (Qian et al., 1993), and the barley alpha-amylase (Kadziola et al. 1994, Journal of Molecular Biology 239: 104–121, A.Kadziola, Thesis, Dept of Chemistry, U. of Copenhagen, Denmark). Furthermore, the three-dimensional structure of a Bacillus circulans cyclodextrin glycosyltransferase (CGTase) is known (Klein et al., 1992) (Lawson et al., 1994). The CGTase catalyzes the same type of reactions as α-amylases and exhibits some structural resemblance with α-amylases.
Furthermore, crystallization and preliminary X-ray studies of B. subtilis α-amylases have been described (Chang et al. (1992) and Mizuno et al. (1993)). No final B. subtilis structure has been reported. Analogously, the preparation of B. licheniformis α-amylase crystals has been reported (Suzuki et al. (1990), but no subsequent report on X-ray crystallographic analysis or three-dimensional structure are available.
Several research teams have attempted to build three-dimensional structures on the basis of the above known α-amylase structures. For instance, Vihinen et al. (J. Biochem. 107, 267–272, 1990), disclose the modelling (or computer simulation) of a three-dimensional structure of the Bacillus stearothermophilus α-amylase on the basis of the TAKA amylase structure. The model was used to investigate hypothetical structural consequences of various site-directed mutations of the B. stearothermophilus α-amylase. E. A. MacGregor (1987) predicts the presence of α-helices and β-barrels in α-amylases from different sources, including barley, pig pancreas and Bacillus amyloliquefaciens on the basis of the known structure of the A. oryzae TAKA α-amylase and secondary structure predicting algorithms. Furthermore, the possible loops and subsites which may be found to be present in, e.g., the B. amyloliquefaciens α-amylase are predicted (based on a comparison with the A. oryzae sequence and structure).
A. E. MacGregor (Starch/Stärke 45 (1993), No. 7, p. 232–237) presents a review of the relationship between the structure and activity of α-amylase related enzymes.
Hitherto, no three-dimensional structure has been available for the industrially important Bacillus α-amylases (which in the present context are termed “Termamyl-like α-amylases”), including the B. licheniformis, the B. amyloliquefaciens, and the B. stearothermophilus α-amylase.